Scaling Catalytic Contributions of Small Self‐Cleaving Ribozymes

Abstract Nucleolytic ribozymes utilize general acid‐base catalysis to perform phosphodiester cleavage. In most ribozyme classes, a conserved active site guanosine is positioned to act as general base, thereby activating the 2′‐OH group to attack the scissile phosphate (γ‐catalysis). Here, we present an atomic mutagenesis study for the pistol ribozyme class. Strikingly, “general base knockout” by replacement of the guanine N1 atom by carbon results in only 2.7‐fold decreased rate. Therefore, the common view that γ‐catalysis critically depends on the N1 moiety becomes challenged. For pistol ribozymes we found that γ‐catalysis is subordinate in overall catalysis, made up by two other catalytic factors (α and δ). Our approach allows scaling of the different catalytic contributions (α, β, γ, δ) with unprecedented precision and paves the way for a thorough mechanistic understanding of nucleolytic ribozymes with active site guanines.


Supporting Figures
Supporting Figure S1

2'-O-(2-Azidoprpyl)uridine modified solid support (6)
Amino-functionalized solid support (GE Healthcare, Custom Primer Support 200 Amino, 436 mg) was transferred into a syringe equipped with a polypropylene filter. The resin was washed with dry dichloromethane, followed by dry N,N-dimethylformamide. Then, the solid support was suspended and swelled in 2.5 ml N,N-dimethylformamide for 30 minutes. Compound 5 (103 mg, 112 µmol) was dissolved in a small amount of N,N-dimethylformamide. Subsequently the mixture was combined with the resin suspension in the syringe. The suspension was shaken for 48 hours at room temperature. Then, the solvent was filtered off by the syringe and the remaining solid support was washed four times with N,Ndimethylformamide, methanol and dichloromethane, and subsequently allowed to dry. In a final capping step, the resin was treated with a mixture of 3.0 mL Cap A ((acetic anhydride/2,4,6trimethylpyridine/acetonitrile, 2/3/5) and 3.0 mL Cap B (4-(N,N-dimethylamino) pyridine/ acetonitrile, 0.5 M) and was shaken for 4 min at room temperature. Finally, the solid support was washed several times with acetonitrile, methanol and dichloromethane. The product was removed from the syringe and dried under vacuum.

Mass spectrometry
All experiments were performed on a Finnigan LCQ Advantage MAX ion trap instrumentation connected to a Thermo Fisher Ultimate 3000 HPLC system. RNAs were analyzed in the negative-ion mode with a potential of −4 kV applied to the spray needle. Monitoring and purification of the reactions was performed by anion exchange HPLC. In case of incomplete reactions, the reaction mixture was precipitated and the corresponding labeling reaction was repeated without previous HPLC purification steps.

Kinetics of ribozyme cleavage (HPLC assay) 2,3
Nanomole aliquots (2.64 nmol) of the ribozyme and substrate strand were taken from aqueous stock solutions, mixed and lyophilized. The RNA was dissolved in 33.6 µl (78.6 µM) nanopure water, heated to 90 °C for 2 min and allowed to cool to room temperature. For time point 0 (prior to the Mg 2+ induced cleavage reaction) 2.8 µl (220 pmol) of the RNA solution were diluted with nanopure water to a total volume of 100 µl. Further, 6.6 µl HEPES buffer (200mM, pH 7.5) and 2. (1)

Determination of pH-rate profiles for ribozyme cleavage (FRET assay/HPLC assay) 4,5,6
The experimental setup was performed as described above (Kinetics of ribozyme cleavage (FRET assay and HPLC assay, respectively)). The pH series for the ribozyme variants were measured in corresponding buffer systems (Supporting Table S1), with three independent measurements for each pH value. The according pH values were fitted against the observed rate constants (kobs) by using a three-parametric (kmax, pKa A , pKa B ) equation (2), representing a bell-shaped pH profile, revealing a maximum at (pKa A + pKa B )/2. Kmax reflects the maximum of the cleavage activity in the case of complete protonation of the general acid and complete deprotonation of the general base, independent of the pH value. Consequently, pKa A denotes the pKa of the acidic group that is deprotonated and pKa B denotes the pKa of the basic unit that is protonated, respectively. In the presence of an influencer a cubic cooperative model, reflected by equation (3) was applied, including a pKa of the influencer species termed as pKa I , leading to pKa shifts denoted as DpKa coop and two plateaus for the cleavage activity that refer S15 to k1 and k2. As a consequence of the small difference among the apparent pKa values (DpKa~1) the rate-pH profiles remain sharp, limiting the accuracy of the predicted values and errors, respectively. Therefore, we fixed kmax and k1, respectively, to a certain value, that showed the most precise fit for our data points with reliable errors. Data processing was performed by using the software package OriginPro 2018 (OriginLab, USA).  Table S1. Buffer systems used for cleavage rate-pH profile studies:   [a] c 3 G -3-deazaguanosine, c 1 G -1-deazaguanosine, X -xanthosine, rS -rSpacer, c 1 I -1-deazainosine, [b] Reversed-phase LC-ESI mass spectrometry (see Methods).